Plant protein for nutritional products and method of making same

ABSTRACT

The present invention relates generally to a method of producing plant proteins for use in nutritional products that have reduced levels of phytoestrogens, manganese or nucleotides. More specifically, this invention is directed to a method of using ion exchange technology to remove phytoestrogens, manganese or nucleotides from plant proteins. This invention is also directed to the plant protein product resulting from the inventive process and to nutritional products that use the plant protein product as a source of amino nitrogen.

This application is a divisional of application Ser. No. 08/713,904filed Sep. 13, 1996, now U.S. Pat. No. 5,804,234.

RELATED CASES

This application is related to co-pending application Ser. No.08/710,236, filed Sep. 13, 1996, entitled PROCESS FOR ISOLATINGPHYTOESTROGENS FROM PLANT PROTEIN. The teachings of that patentapplication are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to a method of purifying plantproteins for use in nutritional products that have reduced levels ofphytoestrogens, manganese and nucleic acids. More specifically, thisinvention is directed to a method of using ion exchange technology toremove phytoestrogens, manganese, nucleotides, nucleosides and RNA fromplant proteins. This invention is also directed to the plant proteinproduct resulting from the inventive process and to nutritional productsthat use the plant protein product as a source of amino nitrogen.

BACKGROUND OF THE INVENTION

Phytoestrogens or plant estrogens occur in a variety of plants,including vegetable protein materials such as those derived fromsoybeans. Phytoestrogens are defined as plant substances that arestructurally and functionally similar to the gonadal steroid 17β-estradiol or that produce estrogenic effects. There are three maingroups of nonsteroidial dietary estrogens which are 1) the isoflavones,2) the coumestans and 3) the mycoestrogens (fungal). The structuralsimilarity between these substances and the endogenous mammalianestrogens have been studied. A review of phytoestrogens and theireffects in mammals is reported by Kaldas and Hughes in an articleentitled, "Reproductive and General Metabolic Effects of Phytoestrogensin Mammals", Reproductive Toxicology, Vol. 3, pp. 81-89, 1989. Theteachings of this article are herein incorporated by reference. As usedin this specification and the appended claims, the term "isoflavones" isequivalent to the term "phytoestrogens" as that term is defined in theKaldas et al. article. Representative of the isoflavones that arereduced in plant proteins in accordance with the present invention aredaidzein, daidzin, genistein and genistin.

Flavonoids and isoflavones are produced by numerous leguminosoe andgrasses, including many plants commonly consumed by man and livestock.Soy isoflavones include compounds such as daidzin, genistin, daidzeinand genistein. A general structural formula for these compounds is:##STR1##

It has recently been recognized that isoflavones contained in vegetableproteins may have a detrimental impact upon the mammals that consume thevegetable protein, see Kaldas et al., supra. The concentration ofisoflavones in plant protein isolates or concentrates such as soyprotein isolates, can be as high as 3,000 μg/g of protein. Isoflavonesalso provide the bitter or "beany" taste to vegetable proteins, (seeEwan et al. infra) may reduce the bioavailability of essential mineralsand may influence the nutritional value of proteins (see Kaldas et al.,supra). The consumption of isoflavones by man and livestock has alsobeen connected with compromised reproductive systems in mammals. Thereis some concern that consumption of current soy based infant formulasthat contain soy isoflavones may have an undesired physiological impacton the developing neuro-endocrine system of the infant. This concern isbased in part, on evidence that soy-based animal feed may causefertility problems in cheetahs. Setchell et al., 1987:"Gastroenterology" 93:225-33.

Further, the presence of high levels of manganese in body tissues hasbeen suspected in the development of criminal behavior. See Gottschalket al., "Abnormalities in Hair Trace Elements as Indicators of AberrantBehavior", Compr Psychiatry 1991; 32:229-237, and Scientific American,March, 1995 pp. 104-105. Furthermore, there have also been reports thatlearning disabilities in children may be associated with increasedlevels of manganese in hair as reported by Collipp et al., in an articleentitled, "Manganese in Infant Formula and Learning Disabilities", Ann.Nutritional Metals, 27:488-494, 1983. Typical plant protein isolatescontain up to 1000 μg of manganese per gram of protein. Thus, there is aneed for improved processes that economically and on a commercial scale,provide for the reduction of isoflavone and manganese content in plantprotein.

The use of nucleotides and nucleosides (or nucleotide equivalents asdefined below) in nutritional formulas has received much attention inthe last few years. It has been suggested that certain levels and ratiosof the various nucleic acids can have a positive impact on the mammalianimmune system and even prevent certain maladies such as diarrhea. Theproblem with using plant protein in such nutritional formulas is thatthe plant protein contains typically very high, inherent level ofnucleic acids that may not be in the correct form (i.e., RNA) and at thecorrect ratios. Further, the high level of variation in the nucleic acidcontent causes problems in commercial manufacture. Typical plant proteinisolates contain up to about 15 mg of nucleotide equivalents per gram ofprotein. Thus, the nutritional industry desires a source of plantprotein that has substantially reduced levels of inherent nucleic acids.One additional benefit to the process of this invention is that, notonly can the isoflavones and manganese be removed by the ion exchangecolumn but also a substantial portion of the inherent nucleic acids.

Ion-exchange technology has been known for a great number of years.Ion-exchange resins are typically synthetic, insoluble, cross-linkedpolymers carrying acidic or basic side groups. They have high exchangecapacities and can be used for an almost unlimited number of reactions.Ion-exchange resins are used in water-treatment, extraction, separation,analysis and catalysis.

Ion-exchange resins have an extended, open molecular framework thatincludes electrically charged ionic groups. A cation exchanger exchangespositive ions and therefore has negative ions built into its framework.An anion exchanger has positive ions in its framework. The ions of thelattice are called the fixed ions; the smaller ions of opposite chargethat can change places with ions in the solution are called counterions.

Common problems encountered with ion exchange processes conducted onproteins include poor protein recovery (i.e., protein adhered to theresin) and inability of the protein slurry to pass through the resin bedresulting in a high pressure drop across the resin bed. The processwhich is disclosed herein fulfills the need in the nutritional industryfor a source of plant protein that has highly reduced levels ofisoflavones, manganese and nucleotides is economical, provides goodprotein recovery and can be used on a commercial scale.

U.S. Pat. No. 5,352,384 to Shen discloses a process to produce anisoflavone enriched vegetable protein fiber. This patent discloses theuse of a glucosidase to convert the glucone isoflavones (i.e., daidzen)in a protein slurry to the aglucone isoflavones. The fiber fraction isthen recovered from the slurry by centrifugation to provide an agluconeenriched fiber.

An article by Ewan et al. in the Journal of Food Science, Vol. 57, No.2, 1992 entitled: "Isoflavone Aglucones and Volatile Organic Compoundsin Soybeans; Effects of Soaking Treatments", discloses the beneficialeffects of soaking soybeans in mildly alkaline NaHCO₃ solutions atelevated temperatures, for manufacturing soymilk with improved flavor.This publication does not suggest or disclose the use of an ion-exchangeresin to remove isoflavones, manganese and nucleic acids from plantprotein.

In an article published in volume 47 (1982) of the Journal of FoodScience, pp. 933-940, by J. How and C. Morr entitled "Removal ofPhenolic Compounds from Soy Protein Extracts Using Activated Carbon",they report subjecting soy protein extracts to activated carbon and ionexchange process treatments to remove phenolic compounds that have beenreported as being responsible for adverse color and flavorcharacteristics of soy protein products. Protein extracts were subjectedto a two stage, sequential ion exchange treatment prior to proteinprecipitation. The protein extract was pumped "down-flow" through acation exchange column in the Na+form and then an anion exchanger in thehydroxyl and chloride form to remove polyvalent anions includingphenolic acids, phytate and others.

U.S. Pat. No. 5,248,804 to Nardelli et al. discloses a process for theremoval of phytate from plant protein using ion-exchange resins. Theprocess uses a macroporous anion exchange resin (weak base or strongbase) which has been conditioned by 1) conversion to the hydroxide form;2) conversion to the chloride or sulfate form; and 3) thereafterconversion of the strong base sites to the carbonate form and the weakbase sites to the free base form. The plant protein containing phytateis then contacted with the treated resin to remove the phytate. Theteachings of U.S. Pat. No. 5,248,804 are herein incorporated byreference.

Phytate comprises the salts of phytic acid. Phytic acid is also known asinositol hexaphosphate. Thus, in using an anion exchange resin, thehighly anionic phosphate groups provide the handle by which the resincan extract the phytate from the protein slurry. In contrast,isoflavones and nucleotides are neutral molecules and would not beexpected to attach to the resin or exchange with the anions on theresin.

U.S. Pat. No. 5,492,899 to Masor et al. discloses an infant formula withribo-nucleotides. This patent teaches the use of certain levels andratios of nucleotide equivalents in infant formulas and discloses ananalytical technique to identify and quantify the nucleotide equivalentsin a nutritional matrix. As used herein and in the claims of thisinvention, the term "nucleotide" is the same as the term "nucleotideequivalent" as defined in U.S. Pat. No. 5,492,899. U.S. Pat. No.5,492,899 defines nucleotide equivalents as polymeric RNA,ribo-nucleosides, ribo-nucleosides containing adducts and mono-, di- andtriphosphate ribonucleotides. The teachings of U.S. Pat. No. 5,392,899are herein incorporated by reference.

The present invention comprises a method through which low isoflavone,low manganese or low nucleotide plant proteins can be manufactured. Theinvention further comprises the low isoflavone, low manganese and lownucleotides protein isolates themselves and to such protein isolatesthat are produced according to the method of the present invention. Thepresent invention further comprises nutritional products made with theprotein isolates produced in accordance with the invention. This, andother aspects of the invention are specifically described in detail inthe description set forth below.

SUMMARY OF THE INVENTION

In its broadest application, the present invention relates to a methodof reducing the isoflavone, manganese or nucleotide content of a plantprotein comprising:

a) providing at least one anion exchange resin;

b) providing a slurry of plant protein that contains isoflavones,manganese or nucleotides;

c) contacting said slurry with said anion exchange resin; and

d) separating said slurry with reduced content of isoflavones, manganeseor nucleotides from said anion exchange resin.

Representative counterions useful in the anion exchange resin accordingto this invention, include acetate, citrate, chloride, bisulfate,carbonate and bicarbonate. As most anion exchange resins are supplied inthe chloride form, it is possible to use such chloride resins directlywithout pretreatment. As discussed below, a preferred procedure forresin pretreatment washes the chloride resin with caustic to clean theresin, then a HCl wash is conducted to clean and control microbialgrowth and then the resin is converted to the carbonate and/orbicarbonate form.

In the production of plant protein using the process according to thisinvention, the anion that is released from the resin as a result ofentrapping the isoflavone, manganese or nucleotide is important to thequality of the finished product. That is to say, the resulting proteinshould not be denatured, contain unacceptable levels of free hydroxylgroups or other offensive anions (i.e., chloride) that would produce aprotein product that would be unacceptable for use in a nutritionalproduct. For example, typical soy protein isolate contains sufficientlevels of isoflavones, manganese and nucleotides that treatment with ananion exchange resin that has chloride as the counterion would produce aresulting protein that has excessive levels of chloride. In similarfashion, if the counterion is hydroxyl, the resulting product would needto be treated with acid to neutralize the basic product, thusunacceptably increasing the mineral load associated with the protein.

In one preferred embodiment of this invention, the anion exchange resinuses a counterion, such as carbonate or bicarbonate, which avoids theaforementioned problems. As used in the specification and in theappended claims, the term "carbonate" means carbonate and bicarbonate.

There is disclosed a method of reducing the isoflavone, manganese ornucleotide content of plant protein comprising:

a) providing at least one anion exchange resin containing strong basesites and weak base sites, said anion exchange resin prior to step b),being subjected to the steps comprising:

i) conversion to a hydroxide form;

ii) conversion to a chloride or sulfate form; and

iii) conversion of at least some of said strong base sites to thecarbonate form and at least some of said weak base sites to the freebase form;

b) providing a slurry of plant protein;

c) contacting said slurry with said anion exchange resin; and

d) separating said slurry with reduced content of isoflavones, manganeseor nucleotides from said anion exchange resin.

There is further disclosed a method for separating compounds selectedfrom isoflavone, manganese or nucleotides from a plant protein slurry,said method comprising the steps of:

a) selecting an anion exchange resin;

b) exposing the resin to an agent that places on the resin anexchangeable anion that:

i) does not change the pH of the protein slurry outside the range of 6.0to 9.5; and

ii) does not add an objectionable anion to the effluent protein slurryat step d);

c) providing a slurry containing a source of plant protein and at leastone compound selected from isoflavone, manganese and nucleotides;

d) bringing the resin into contact with the resin; and

e) separating the slurry from the resin.

The present invention also relates to the protein that results from theprocess described herein. The protein product or isolate according tothis invention is characterized in that it contains less than 30 μg ofisoflavones per g of protein, less than 450 μg of manganese per g ofprotein and less than 10 mg of nucleotides per g of protein. The presentinvention further relates to a plant protein composition which comprisesless than 30 μg of isoflavones per g of plant protein and to nutritionalproducts comprising said protein.

There is also disclosed infant formulas that are based on plant proteinand contain less than 600 μg of isoflavones per liter of ready-to-feedformula, more preferably less than 200 μg and most preferably less than100 μg.

DETAILED DESCRIPTION

Typically, the method of this invention is conducted by placing theanion exchange resin in a bed, column or reactor through which theprotein slurry is passed. The bed, column or reactor has at least oneinlet and at least one outlet and is preferably operated as a verticalcolumn in the "upflow" mode. In another embodiment, the preconditionedresin may be added to a tank containing the protein slurry and after anappropriate period of time for the reaction to take place, the resin isfiltered from the slurry.

The anion exchange resin is typically a macroporous resin and ispreferably a Type I or II macroporous resin. In a preferred embodiment,the anion exchange resin is selected from weak base anion exchangeresins, strong base anion exchange resins and mixtures thereof.Representative of the anion exchange resins useful in the presentinvention include Amberlite® RA95, IRA-910 and IRA-900 sold by Rohm andHaas Company, Dowex-22 and MSA-1 sold by Dow Chemical and Purolite A510and A500 sold by Purolite Company. As used herein and in the claims, theterm resin is meant to include gels, which those skilled in the artwould understand to be useful in the process described herein.Representative of such gels are Amberlite® IRA 410 (Type II gel, strongbase anion) sold by Rohm and Haas Company, IRA 402 is a Type I strongbase anion exchange gel that is not macroporous that would also beuseful.

The proteins that may be used in the method of this invention includeany plant protein that contains detectable levels of isoflavones,manganese and nucleotides. More specifically, the protein is obtainablefrom soybeans, corn, wheat, peas, beans, cottonseed, peanuts, carrots,alfalfa, apples, barley, bluegrass, clovers, coffee, garlic, hops,marijuana, oats, algae, orchard grass, parsley, rice, rye, sage, sesame,yeast, fungus, potatoes, hydrolyzates thereof and mixtures thereof.

It is preferred that the protein be a protein isolate or concentratewherein the levels of fats, oils and carbohydrates have been reduced. Ithas been determined that the presence of fats and oils reduces theefficiency of the inventive process.

The chemical agents useful in converting the resin to the hydroxide forminclude sodium hydroxide, calcium hydroxide, potassium hydroxide andmagnesium hydroxide. The most preferred agent is sodium hydroxide.

The chemical agents useful in converting the resin to the chloride orsulfate form include hydrochloric acid, sulfuric acid and sodiumchloride. The preferred agent is hydrochloric acid.

The chemical agents useful in converting the resin to the carbonate orfree base form include any of the weak base salts such as sodiumcarbonate, sodium bicarbonate and ammonium hydroxide. Sodium bicarbonateis the most preferred agent as it produces a protein effluent at a pHrange of 6.6-9.5.

Those skilled in the art of ion exchange technology will appreciate thatthe protein slurry containing the isoflavones, manganese or nucleotides,as it is contacted with the anion exchange resin, should be at a pH thatdoes not denature the protein, which causes clogging of the column.Further, adjustment of the pH past neutral, will add significant levelsof anions to the slurry which will compete for counterion sites.Typically, a pH of from about 5.5 to 10 is satisfactory. Preferably, thepH of the protein slurry feed can range from 6.0 to 8.0. The pH of theprotein slurry effluent (leaving the column or bed) should be near thepH at which the protein will be used in a final product. Thus, if aplant protein treated in accordance with this invention is to be used inan infant formula, the effluent pH should be about 6.0 to 7.5. In apreferred embodiment, the plant protein feed to the resin should be asfree of added anions (i.e., --OH, Cl--, and the like) as possible. Theaddition of acids, bases, salts and the like to the protein slurry feeddecreases the efficiency of the column to remove the isoflavones,manganese or nucleotides from the protein slurry.

As those skilled in the art will appreciate, exchange resins have afinite capacity and may be regenerated to an active state afterexhaustion or near exhaustion. Thus, as contemplated in this invention,the exchange resins after contact with the plant protein is regeneratedor reconditioned through known steps to the anionic form or morepreferably through the steps comprising:

1) stripping the resin of any residue (i.e., protein) and conversion tothe hydroxide form, for example through the use of sodium hydroxide;

2) conversion of the resin to the chloride or sulfate form; and

3) conversion of the strong base sites on the resin to the carbonateform and conversion of the weak base sites to the free base form.

Those skilled in the ion exchange resin art will appreciate thatnon-aqueous and alcohol water regenerations can be used.

One preferred embodiment of the method according to the presentinvention includes the step of homogenizing the plant protein slurryprior to contact with the resin. Homogenization or treatments similarthereto have been found in the process of this invention, to increasethe effective removal of isoflavones, manganese and nucleotides from theslurry. In addition, homogenization of the protein slurry prior tocontact with the resin reduces the pressure drop across the resin bed orcolumn which facilitates the facile and economic production of a plantprotein for use in nutritional products.

The present invention is also directed to a plant protein isolate thathas specified levels of isoflavones and to a plant protein that has beensubjected to the process disclosed herein and to nutritional productsthat are made from such proteins. Also contemplated herein are animalfeeds that are substantially free of isoflavones. More specifically, thepresent invention relates to a plant protein containing less than about30 μg of isoflavones per g of protein, less than about 450 μg ofmanganese and less than about 10 mg of nucleotide equivalents per gramof protein. In a more preferred embodiment, the protein is derived fromsoy beans and contains less than 20 μg isoflavone per gm of protein. Ina most preferred embodiment, the plant protein contains less than 10 μgisoflavone per gm of protein, less than 5 mg of nucleotides per gm ofprotein and less than 200 μg of manganese per g of protein.

The following Examples describe specific, but non-limiting, embodimentsof the present invention. The features of the present invention whichare believed to be novel are set forth with particularity in theappended claims and should be understood as to structure and manner ofoperation by the following detailed Examples.

EXAMPLE 1

The process according to the present invention was used to produce atotal of 221 kg (487 lbs.) of low isoflavone, low manganese and lownucleotide content soy isolate powder that was used in the manufactureof an infant formula. A total of six (6) manufacturing runs wererequired to produce the required soy protein isolate. A substantialamount of experimentation was conducted on a 50 liter scale to result inthe best mode, described herein.

The soy protein starting material used in this Example was obtained fromArcher Daniels Midland, Inc. (ADM) of Decatur, Ill., in curd form. Thecurd or protein slurry was of the commercially available soy proteinisolate product known as Ardex F®. In a typical commercial process, soyproteins are extracted at slightly alkaline pH from defatted soy flakeor defatted soy flour. The protein fraction is then precipitated fromthe extract by adjusting the pH to the isoelectric point of the proteins(pH 3.8 to 6.0). As most of the proteins are insoluble at this pH, acurd forms and the protein curd can be separated from the solublesugars, salts, etc., by centrifugation. To complete the purification,the protein curd is washed with water at least once at this isoelectricpH, then the protein is spray-dried either as is or after resuspensionat a neutral pH. In the following experiments, ADM supplied theisoelectric curd at 10 to 14% total solids and at a pH of about 4.5.

The supplied soy curd was diluted to about 6.5% total solids with waterand placed into steam jacketed kettles. Each batch of theprotein-in-water slurries weighed about 908 kg (2000 lbs.). The slurrywas then heated to about 49° C. (120° F.) and neutralized to a pH of 6.8with NaOH. The slurry was then filtered through a 60 mesh strainer,UHTST (ultra high temperature short time) processed and homogenized. TheUHTST steam injection was at 152° C. (305° F.) and held for 10 seconds.It was determined that UHTST treatment post anion exchange exposureproduces a protein with undesirable organoleptic properties. The slurrywas then cooled to 55° C. (130° F.) and homogenized at 6895 kPa (1000psig). The slurry was then transferred to the ion exchange system.

One aspect of the present invention resides in the discovery that ultrahigh temperature short time (UHTST) treatments need to be conductedprior to contact of the slurry with the resin to prevent spoilage of theslurry during the extended processing times. The process is conducted attemperatures where rapid microbiological growth can occur.Representative of the UHTST conditions useful in the present inventionare temperatures from 120° C. (250° F.) to 155° C. (3 10° F.) and timesof 1 to 60 seconds. The lower temperatures are associated with thelonger hold times. This UHTST treatment, prior to the slurry contactingwith the resin, provides microbiological stability while minimizingnutrient degradation.

The ion exchange system comprised a stainless steel, rubber lined columnhaving inlet and outlet ports and a height of 401 cm (13'2") and adiameter of 30.5 cm (12 inch). 70 liters of Amberlite® IRA-910 anionexchange resin from the Rohm and Haas Co. of Philadelphia, Pa. wasplaced in the column. IRA-9 10 is a macroreticular strongly basic anionexchange resin. The basicity of this resin is derived from quaternaryammonium functionality with slightly lower basic strength than a Type 1anion exchange resin. This resin is supplied in the chloride form and isapproved by the United States Food and Drug Administration (FDA) (aftercondition cycling) for use in processing edible products.

Prior to contact with the protein curd, the resin was preconditioned.The resin was preconditioned through contact in an upflow mode with 6%by weight NaOH at a flow rate of 4.6 to 5.7 liters (1.2 to 1.5 gal) perminute for 30 minutes. The resin bed was then washed with deionizedwater for 10 to 15 minutes in the upflow mode. The resin was thencontacted with 1.0% by weight HCl in a downflow mode at 16 liters perminute. The resin was then washed with deionized water in the downflowmode for about 30 minutes. 2.8 kgs (6.18 lbs) of sodium bicarbonate wasadded to about 196 liters (49 gals) of water and agitated to dissolve.This solution was then pumped to the column in a downflow mode at about4 liters per minute. The bed was again rinsed with deionized water untilthe conductivity of the effluent was 300 μmhos or less. The resin bedwas then backwashed to remove air and re-classify the resin. The resinbed was allowed to settle naturally and the water was drained from thecolumn. The column was now ready for the service cycle after drainingthe water to the top of the resin bed.

The protein slurry was pumped upflow through the ion exchange column ata flow rate of 3.6 to 3.8 kgs (8 to 8.4 lbs.) per minute. The inlettemperature of the slurry was 55-60° C. (130-140° F.) and the contacttime was about 20 minutes minimum. Protein slurry exiting the column wascooled, samples were taken and then spray dried using conventionaltechniques and equipment.

The column before the next batch was regenerated with 6% NaOH, 1% HCland 1.5% NaHCO₃ (sodium bicarbonate) as described above for the initialpreparation of the resin bed. All solutions were prepared with deionizedwater.

Results

A total of six batches were manufactured to yield a total of about 221kgs of ion exchanged soy isolate powder. Three (3) samples were taken atvarious times during the processing of each batch: 1) protein slurry fedto the ion exchange column; 2) effluent from the column; and 3) driedpowder. The samples were analyzed for mineral profiles of sodium,potassium, phosphorus, chlorine, calcium, magnesium, manganese, aluminumand fluoride. The samples were also analyzed for isoflavones andnucleotides. In order to make comparisons between the liquids and thepowders possible, the concentration of the powder was normalized to61/2% total solids.

Mean levels and standard deviations for each analyte before and afterion exchange were calculated for the six runs. The results are set forthin Table I. Note that reduction is expressed as a positive value while anegative value represents an increase in analyte concentration.

                  TABLE I                                                         ______________________________________                                        Mineral Profile                                                               Mineral       Reduction %                                                     ______________________________________                                        Phosphorus    73.3 ± 3.4                                                   Calcium       16.5 ± 4.2                                                   Magnesium     11.4 ± 5.1                                                   Sodium        -6.3 ± 3.9                                                   Potassium     -7 ± 21                                                      Manganese     31 ± 10                                                      Aluminum       6 ± 15                                                      Chloride      -270 ± 110                                                   Fluoride      48 ± 29                                                      ______________________________________                                    

The most significant reduction in concentration from the ion exchangetreatment was observed in total phosphorus, flouride and manganese. Thereduction in phosphorus in consistent with the teachings of U.S. Pat.No. 5,2478,804 as a large portion of the phosphorus inherent in soyexists as a phytate salt. In contrast, the effluent showed a significantincrease in chloride. This is consistent with the fact that HCl is oneof the regenerants used after the caustic rinse and the strong baseresin has some weak base sites.

The profiles of calcium, magnesium, manganese, fluoride and aluminumbefore and after treatment showed a decrease. Of this group, manganeseshowed a significant decrease (31±10%). Surprisingly, when compared tothe other multi-valent metals, aluminum (+3 charge) remained essentiallyunchanged. Further, the removal of calcium and magnesium can beexplained as the adsorption or chelation with phytate.

The monovalent cations, sodium and potassium, were relatively unaffectedby the ion exchange treatment (-6.3±3.9% and -7±21%, respectively). Thenegative values actually indicate a slight uptake in both sodium andpotassium. These data support the typical behavior of an anion exchangeresin in that monovalent cations would not be exchanged or adsorbed bythe anionic resin.

An important benefit of the instant inventive process is that highlevels of protein are recovered from the treated plant protein isolates.This means that very little protein is lost in the resin column or bed.In these experiments, over 90% of the protein that entered the resincolumn was recovered in the effluent.

It is important to note that the overall efficiency of the process ofthis invention is improved when solubility and homogenity of the proteinslurry is enhanced. Thus, the pre-filtration (through a 60 mesh filter)and homogenization greatly reduced the pressure drop across the column,which increased the efficiency of the inventive process. In comparison,the process without the pre-filtering and homogenization resulted in aninitial pressure drop of about 138 kPa (20 psig) while the pre-filteringand homogenization resulted in an initial pressure drop of about 14 to35 kPa (2-5 psig). After about 4 to 6 hours of operation withoutprefiltering and homogenization, the pressure drops of 276 to 414 kPa(40-60 psig) were experienced while with prefiltering andhomogenization, pressure drops were about 55 to 83 kPa (8-12 psig).

The process of this invention was also very effective in removingnucleotides. The analytical procedures used are described in U.S. Pat.No. 5,492,899 to Masor et al. The removal of total potentially availablenucleotides (TPAN) was found to be about 57.4±7.2%.

The isoflavones were almost completely removed by the present inventiveprocess. Table II sets forth the specific isoflavones, the level of thefeed slurry, level in the effluent and level in the powder.

                                      TABLE II                                    __________________________________________________________________________                         REDUCTION %                                                     FEED   EFFLUENT                                                                             POWDER FEED VS.                                                                           FEED VS.                                     ISOFLAVONE                                                                           μg/g*                                                                             μg/g*                                                                             μg/g*                                                                             POWDER                                                                             EFFLUENT                                     __________________________________________________________________________    Daidzin                                                                              4.12 ± 0.87                                                                       0.51 ± 0.21                                                                       0.68 ± 0.37                                                                       83.5 ± 6.8                                                                      87.6                                         Genistin                                                                             10.0 ± 2.8                                                                        0.82 ± 0.55                                                                       0.87 ± 0.72                                                                       91.4 ± 5.9                                                                      91.8                                         Daidzein                                                                             3.9 ± 6.5                                                                         0.10 ± 0.0                                                                        0.10 ± 0.0                                                                        97.4 ± 4.3                                                                      97.4                                         Genistein                                                                            3.7 ± 1.4                                                                         0.10 ± 0.0                                                                        0.10 ± 0.0                                                                        97.3 ± 1.4                                                                      97.3                                         __________________________________________________________________________     *normalized to a 6.5 wt % solution                                       

EXAMPLE II

Nutritional Product Using Low Isoflavone Soy Protein

The soy protein produced in Example I was used to produce an infantformula. A control product and the infant formula according to thisinvention were then analyzed for isoflavone content. The procedure usedto produce the experimental and control products was that described inU.S. Pat. No. 5,021,245 to Borschel et al. except that the fiber wasomitted. The teachings of U.S. Pat. No. 5,021,245 are hereinincorporated by reference.

Typically, plant protein based infant formulas contain 1.5 to 2.0 wt %protein as fed (ready-to-feed). A preferred embodiment is 1.6 to 1.8 wt% protein as fed. Thus, as described below, an infant formula made witha plant protein treated in accordance with this invention will have anisoflavone content of less than 600 μg/liter of formula. (30 μg ofisoflavone per g of protein x 20 gms protein per liter of formula(RTF)=600 μg if isoflavone per liter of RTF formnula). A more preferredinfant formula according to this invention will contain less than 200 μgof isoflavones per liter of RTF formula and most preferably less than100 μg of isoflavones per liter of RTF formula.

A further aspect of the invention is directed to a soy proteincontaining 43.6 micrograms of isoflavone per gram of protein in whichsaid isoflavones are selected from the group consisting of daidzin,genistin, daidzein, and genistein. An additional aspect of the inventionis directed to an infant formula containing 1092 micrograms, or less, ofisoflavone per liter of formula in which said isoflavones are selectedfrom the group consisting of daidzin, genistin,daidzein and genistein. Afurther aspect of the invention is directed to a formula containing 1526micrograms or less, of isoflavone per liter of formula in which saidisoflavones are selected from the group consisting of daidzin, genistin,daidzein, and genistein.

An HPLC (high pressure liquid chromatography) method was used toquantitate the major soy isoflavones (genistin, daidzin, genistein anddaidzein) using a method adapted from the following three (3) articleswhich are herein incorporated by reference.

1) Setchell, K D R and Welch, M B J. Chrom. 386 (1987) 315-323

2) Wang, G., Kuian, S S, Francis, O J, Ware, G M, and Corman, A S J.Agric. Food Chem. 38 (1990) 185-190

3) Barnes, S., Kirk M., and Coward, L. J. Agric. Food. Chem. 42 (1994)2466-2474

Samples of the ready-to-feed experimental and control infant formulawere obtained and 20 ml of each was weighed into a tared 250 ml roundbottom flask. 80 ml of ethyl alcohol was then added and the mixture wasstirred. A condenser was attached to the flask and the samples wererefluxed at 80° C. for 2 hours. The mixtures were then cooled to roomtemperature and quantitatively transferred to a 100 ml volumetric flask.The precipitate and the flask were rinsed with 15 ml of 80% alcohol(v/v). The volumetric flasks were brought to volume with 80% alcohol andthe samples were then mixed well. The samples were filtered throughWhatman No. 41 paper and then 15 ml of each filtrate was placed into agraduated conical 15 ml stoppered glass test tube. Each tube was placedin a warm water bath and a stream of nitrogen was used to evaporate eachsample to 3 ml. The tubes were then cooled to room temperature and 1 mlof methanol was added to each tube and then diluted to 10 ml with waterand mixed well. 1.5 ml of each sample was then filtered through a 0.45μm polypropylene membrane into an HPLC autosampler vial. Test analysisfor isoflavones using reverse phase HPLC was conducted with the HPLCsystem as follows:

    ______________________________________                                        Column     Vydac C18 Pharmaceutical; 250 × 4.6 mm; 5 μm              Detection  UV absorbance at 254/280 nm                                        Injection  50 mcL                                                             Temperature                                                                              ambient                                                            Flow Rate  0.8 ml/min                                                         Run Time   120 minutes                                                        Eluant A   950 volumes water; 50 volumes CH.sub.3 CN; 1 volume                           trifloroacetic acid (TFA)                                          Eluant B   400 volumes water; 600 volumes CH.sub.3 CN; 1 volume                          TFA                                                                Gradient Program:                                                             Time (minutes)                                                                           0     5        95  100    102  120                                 % Eluant B 0     0        60  100    100   0                                  ______________________________________                                    

The results of the test are set forth in Table III

                  TABLE III                                                       ______________________________________                                                        Control Experimental                                          Soy Isoflavones μg/g μg/g*                                              ______________________________________                                        Daidzin         11.6    <1.0                                                  Daidzein         1.0    <1.0                                                  Genistin        19.4    <1.0                                                  Genistein        2.2    <1.0                                                  TOTAL           34.2    <1.0 N/A                                              ______________________________________                                         *At detection limit.                                                     

This indicates that the process of the invention can be used to producea nutritional product that has greatly reduced levels of isoflavones.

These nutritional products include infant formula, said formulacomprising: 1) protein, said protein being of a concentration of between10 and 25 grams per liter of formula; 2) fat, said fat being of aconcentration of between 20 and 45 grams per liter; and 3)carbohydrates, said carbohydrates being of a concentration of between 60and 110 grams per liter of formula. Preferably, the protein has as itssource soy protein isolate, or sodium and calcium caseinates or a blendthereof; said fat has as its source soy, coconut or corn oil, or anothervegetable oil or a blend thereof; and said carbohydrates have as theirsource, sucrose, corn syrup, glucose polymers, or other carbohydrates,or a blend thereof.

The pediatric nutritional formula of this invention is preferablyprepared using the following method. An appropriate quantity of proteinis dispersed in sufficient water to solubilize it, thereby forming aprotein solution. Typically this protein source would be soy proteinisolate. A carbohydrate source such as one or more of corn syrup solids,maltodextrins, and sucrose is dissolved in water, thereby forming acarbohydrate solution. Appropriate minerals are dissolved in water, soas to form a mineral solution. Once formed the solutions (protein,carbohydrate, and mineral) are combined in appropriate quantities withvegetable oils and oil soluble vitamins. The resulting solution is thenheat processed and homogenized. Following processing, water solublevitamins are added. The solution is then diluted with water to theappropriate caloric density, approximately 670-725 kcal per liter offormula. The formula is then dispensed into containers and retorted toobtain commercial sterility. As prepared, the formula containsappropriate nutrients in compliance with the Infant Formula Act as ofthe date of this application. It should also be recognized that theunique formula of this invention could be prepared for use in powderedform or as a concentrated liquid.

A further aspect of this invention is directed to an enteral formulacomprising protein, said protein being of a concentration of from 10 to35 grams per liter; fat, said fat being of a concentration of from 20-45grams per liter; and carbohydrates, said carbohydrates being of aconcentration of from 60-110 grams per liter.

EXAMPLE III Tolerance Study

At the time of filing this application, a clinical study of thephysiological effects of plant estrogens or isoflavones in inflantformula was underway. Prior to this extensive study, a smaller tolerancestudy was conducted to assess overall tolerance of reduced isoflavonesoy formulas in healthy, term infants.

The tolerance study was a randomized, double-masked, 3 week study using145 healthy infants 2 to 5 weeks of age. Infants were fed a standardmilk-based formula for a 1 week baseline period and then fed for 2 weeksa standard soy formula, a low phytate soy isolate based formula withisoflavones, a hydrolyzed soy isolate based formula with reduced phytateand isoflavones or a formula using the protein produced in Example I.The primary outcome variables were stool characteristics, formula intakeand incidence of spit up and vomiting. Secondary variables were weightgain and parental responses to a feeding tolerance questionnaire.

Formula intake and incidence of spit up and vomit did not differ amongthe groups at baseline or during the study period. Mean stool rankconsistency was softer for infants fed the hydrolyzed formula comparedto the other experimentals. Parents associated watery and more frequentstools with the hydrolyzed formula. Infants fed the formula using theprotein produced in Example I evidenced less constipation than atbaseline. Mean weight gains were similar for all study groups. Theconclusion of this study was that removal of phytate and/or isoflavonesfrom the soy based formula had minimal impact of tolerance.

Industrial Applicability

The process disclosed in this invention is a very effective, inexpensiveand reliable method for the commercial removal of isoflavones, manganeseand nucleotides from plant proteins. The process produces a plantprotein that has highly desirable characteristics such as isoflavonecontent of less than 30 μg/g protein, less than 450 μg manganese per gof protein and less than 10 mg nucleotides per g of protein. The proteinresulting from treatment with the process described herein also hasbetter flavor (less beany taste), improved color (lighter) and improvedfunctionality (i.e., ability to form a stable emulsion).

Commercial scale use of the process of this invention is enhanced whenthe protein slurry is pre-filtered and homogenized prior to contact withthe resin bed. Macroporous resins in the bicarbonate form are mostpreferably used.

As a result of the present inventor's advancement to the state of theart, the nutritional industry will now be able to economically produceproducts containing reduced levels of isoflavones, manganese andnucleotides. Lastly, humans and animals consuming products producedaccording to this invention will benefit from the avoidance of certainnoxious elements contained in plant proteins.

While certain representative embodiments and details have been presentedfor the purpose of illustrating the invention, it will be apparent tothose skilled in the art that various changes and modifications may bemade therein without departing from the spirit or scope of theinvention.

We claim:
 1. An infant formula comprising soy protein, carbohydrates,fats, vitamins, and minerals wherein said infant formula contains lessthat 600 micrograms of isoflavones per liter of ready-to-feed formula.2. The infant formula according to claim 1 wherein said formula containsless than 200 μg of isoflavones per liter of ready-to-feed formulas. 3.The infant formula according to claim 2 wherein said formula containsless than 100 μg of isoflavones per liter of ready-to-feed formulas. 4.An infant formula comprising:a) soy protein, said protein being of aconcentration of between 10 and 25 grams per liter of formula; b) asource of carbohydrate; c) a source of fat; d) a source of minerals; e)a source of vitamins, and; f) said infant formula contains less than 600micrograms of isoflavones per liter of formula.
 5. The infant formulaaccording to claim 4 in which said protein is soy protein isolate. 6.The infant formula according to claim 4 in which said carbohydrate ispresent at a level of between 60 and 110 grams per liter.
 7. The infantformula according to claim 6 in which said carbohydrate source comprisesat least one of corn syrup solids, maltodextrins, glucose polymers, orsucrose.
 8. The infant formula according to claim 4 in which said fat ispresent at a level of between 20 and 45 grams per liter of formula. 9.The infant formula according to claim 8 in which said fat sourcecomprises at least one of soy oil, coconut oil, or corn oil.
 10. Theinfant formula according to any one of claims 4, 5, 6, 7, 8, or 9 inwhich said isoflavone is present at a concentration of less than 200micrograms of isoflavones per liter of said formula.
 11. The infantformula according to anyone of claims 4, 5, 6, 7, 8, or 9 in which saidisoflavone is present at a concentration of less than 100 micrograms ofisoflavones per liter of said formula.
 12. The infant formula accordingto anyone of claims 4, 5, 6, 7, 8 or 9, in which said infant formula hasa caloric density of approximately 670-725 kcal per liter of saidformula.
 13. The infant formula according to claim 4 which is in theform of a powdered composition which is diluted prior to ingestion bysaid infant.
 14. The infant formula according to claim 4 which is in theform of a concentrated liquid that is diluted prior to ingestion by saidinfant.
 15. An enteral formula comprising:a) soy protein, said proteinbeing of a concentration of between 10 and 35 grams per liter offormula; b) optionally a source of carbohydrate; c) optionally a sourceof fat; and e) said enteral formula contains less than 600 micrograms ofisoflavones per liter of formula.
 16. The enteral formula according toclaim 15 in which said protein is soy protein isolate.
 17. The enteralformula according to claim 15 in which said carbohydrate is present at alevel of between 60 and 110 grams per liter.
 18. The enteral formulaaccording to claim 17 in which said carbohydrate source comprises atleast one of corn syrup solids, maltodextrins, glucose polymers, orsucrose.
 19. The enteral formula according to claim 15 in which said fatis present at a level of between 20 and 45 grams per liter of formula.20. The enteral formula according to claim 19 in which said fatcomprises at least one of soy oil, coconut oil, or corn oil.
 21. Theenteral formula according to any one of claims 15, 16, 17 18, 19, or 20in which said isoflavone is present at a concentration of less than 200micrograms of isoflavones per liter of said formula.
 22. The enteralformula according to any one of claims 15, 16, 17, 18, 19, or 20 inwhich said isoflavone is present at a concentration of less than 100 μgmicrograms of isoflavones per liter of said formula.
 23. A formulacomprising:a) soy protein, said protein being of a concentration ofbetween 10 and 25 grams per liter of formula; b) a source ofcarbohydrate; c) a source of fat; d) a source of minerals; e) a sourceof vitamins, and; f) said formula contains 1092 micrograms, or less, ofisoflavones per liter of formula, in which said isoflavones are selectedfrom the group consisting of daidzin, genistin, daidzein, and genistein.24. The formula according to claim 23 in which said carbohydrate ispresent at a level of between 60 and 110 grams per liter.
 25. Theformula according to claim 24 in which said carbohydrate sourcecomprises at least one of corn syrup solids, maltodextrins, glucosepolymers, or sucrose.
 26. The formula according to claim 23 in whichsaid fat is present at a level of between 20 and 45 grams per liter offormula.
 27. The formula according to claim 26 in which said fat sourcecomprises at least one of soy oil, coconut oil, or corn oil.
 28. Theformula according to claim 23 in which said formula has a caloricdensity of approximately 670-725 kcal per liter of said formula.
 29. Theformula according to claim 23 which is in the form of a powderedcomposition which is diluted prior to ingestion.
 30. The formulaaccording to claim 23 which is in the form of a concentrated liquid thatis diluted prior to ingestion.
 31. The formula according to claim 23 inwhich said protein is soy protein isolate.
 32. An enteral formulacomprising:a) soy protein, said protein being of a concentration ofbetween 10 and 35 grams per liter of formula; b) optionally a source ofcarbohydrate; c) optionally a source of fat; and e) said enteral formulacontains 1526 micrograms, or less, of isoflavones per liter of formulain which said isoflavones are selected from the group consisting ofdaidzin, genistin, daidzein, and genistein.
 33. The enteral formulaaccording to claim 32 in which said protein is soy protein isolate. 34.The enteral formula according to claim 32 in which said carbohydrate ispresent at a level of between 60 and 110 grams per liter.
 35. Theenteral formula according to claim 34 in which said carbohydrate sourcecomprises at least one of corn syrup solids, maltodextrins, glucosepolymers, or sucrose.
 36. The enteral formula according to claim 32 inwhich said fat is present at a level of between 20 and 45 grams perliter of formula.
 37. The enteral formula according to claim 36 in whichsaid fat comprises at least one of soy oil, coconut oil, or corn oil.38. The formula according to claim 32 in which said formula has acaloric density of approximately 670-725 kcal per liter of said formula.39. The formula according to claim 32 which is in the form of a powderedcomposition which is diluted prior to ingestion.
 40. The formulaaccording to claim 32 which is in the form of a concentrated liquid thatis diluted prior to ingestion.